The G12 family of heterotrimeric G proteins, which consists of G12 and G13, has been shown to mediate signals from GPCRs to RhoA activation. The monomeric GTPase RhoA modulates cellular processes, such as actin cytoskeletal rearrangement and gene expression. RGS-RhoGEF, specific GEFs (guanine nucleotide exchange factor) for RhoA with amino terminal RGS domain, acts as a direct link between G12/13 and RhoA. Studies have demonstrated the importance of G12/13-RGS-RhoGEF signaling pathway in a variety physiological functions. The involvement of this signaling pathway in disease conditions, such as cardiac hypertrophy, immunological dysfunction, or cancer metastasis has also been demonstrated. The long term goal of this proposal is to understand the molecular mechanism and function of heterotrimeric G protein-mediated regulation of Rho family GTPases. Immediate goal of this proposal is to elucidate the molecular mechanism and function of G12/13-RhoGEF signaling pathway. During the current funding period, we have made following progresses: (1) We developed a novel purification method for G12 and G13 and determined their crystal structures as well as G13-p115RhoGEF RGS complex. (2) We successfully applied similar purification strategy for Gq and determined the crystal structure of Gq-GRK2-Gbetagamma complex. Also, we discovered a novel RhoGEF, p63RhoGEF as a direct effector for Gq. (3) Using surface plasmon resonance (SPR) we established a system to measure kinetics and thermodynamics of G13-LARG interaction. The thermodynamic parameters suggested a significant conformational change of LARG during interaction with G13. (4) We discovered that G13 directly interacts with the cytoplasmic domain of the beta3andbeta integrin, and this interaction is required in mediating the integrin outside-in signaling leading to platelet spreading. Based on these progresses, we will investigate the following aims in this proposal. (1) We will determine X-ray crystal structures of the complex of G12 or G13 with p115RhoGEF construct including RGS domain and DH/PH domains. We will also perform SPR analysis to understand the dynamics of G12/13-p115RhoGEF interaction. (2) We hypothesize that phosphorylation of RGS-RhoGEF plays crucial regulatory role in G12/13-mediated signaling. With the combination of specific moloclonal antibodies and mass spectrometry, we will determine the phosphorylation sites of endogenous RGS-RhoGEF and characterize the functional role of these phosphorylation modifications in G12/13- mediated signal transduction. (3) We hypothesize that the signal coordination of G12/13-pathway and integrin-pathway is critical for cell invasion and migration of cancer cells. We will analyze the mechanism of this signal network using X-ray crystallography, reconstitution assays, and biosensor to monitor RhoA activity, and migration and invasion of cancer cells. PUBLIC HEALTH RELEVANCE: G protein-mediated signaling is the most widely used molecular mechanism to control a variety of physiological functions of our body. G protein-coupled receptors (GPCR) consist of the largest gene family of our genome and the most common drug target family. About 50% of current drugs on the market target GPCRs or their signaling components. Thus, the investigation of G protein- mediated signaling pathway is not only essential to understand our physiology but also critically important to develop novel drugs in future. In this project, I will focus on the G12/13-mediated signaling pathway, a specific G protein signaling pathway that is known to be important for various important cellular processes including cancer progression, cell transformation, cell shape changes, or cell growth and differentiation. Experiments were proposed to elucidate the structure of the signaling components of this pathway and also the dynamic changes of interaction between these components. Recently, we also found a novel signaling network between G12/13-signaling pathway and cell adhesion signaling pathway. This is a novel finding to connect these two important signaling systems. The experiments from atomic structure analysis to cellular assay system are proposed to understand this signal network. The results from this project will make crucial contribution to understand advancement of cancer phenotypes and form a solid basis for future anti-cancer drug development.